Functional analysis of the domain structure of tumor necrosis factor-alphaconverting enzyme

Many membrane-bound proteins, including cytokines, receptors, and growth fa ctors, are proteolytically cleaved to release a soluble form of their extra cellular domain. The tumor necrosis factor (TNF)-alpha converting enzyme (T ACE/ADAM-17) is a transmembrane metalloproteinase responsible for the prote olytic release or "shedding" of several cell-surface proteins, including TN F and p75 TNFR. We established a TACE-reconstitution system using TACE-defi cient cells co-transfected with TACE and substrate cDNAs to study TACE func tion and regulation. Using the TACE-reconstitution system, we identified tw o additional substrates of TACE, interleukin (IL)-1R-II and p55 TNFR. Using truncations and chimeric constructs of TACE and another ADAM family member , ADAM-10, we studied the function of the different domains of TACE in thre e shedding activities. We found that TACE must be expressed with its membra ne-anchoring domain for phorbol ester-stimulated shedding of TMF, p75 TNFR, and IL-1R-II, but that the cytoplasmic domain is not required for the shed ding of these substrates. The catalytic domain of ADAM-10 could not be func tionally substituted for that of TACE. IL-1R-II shedding required the cyste ine-rich domain of TACE as well as the catalytic domain, whereas TNF and p7 5 TNFR shedding required only the tethered TACE catalytic domain.